Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
Modern methods of identifying compounds having desired chemical or physical properties typically involve assembling libraries of compounds, which are then systematically screened for members with the desired properties. One method of assembling compound libraries involves the highly labor-intensive process of isolating and characterizing naturally occurring compounds. Another approach involves synthesizing libraries of compounds using combinatorial processes in which sets of compounds are prepared from sets of building blocks via multi-step synthesis. The libraries produced by the latter approach typically successfully emulate the structural characteristics of naturally occurring compounds. In addition, combinatorial libraries also generally provide more rapid access to larger collections of more diverse compounds that may incorporate optimized chemical or physical properties into their structures.
Numerous techniques have been devised for producing combinatorial libraries. Many of these techniques utilize solid supports to exploit efficient xe2x80x9csplit-and-pool,xe2x80x9d or simply xe2x80x9csplit/pool,xe2x80x9d synthesis methods to assemble all possible combinations of a set of building blocks. The split/pool method typically utilizes a pool of solid supports containing reactive moieties. This pool is initially split into a number of individual pools of solid supports. Each pool is then subjected to a first reaction or randomization that results in a different modification to the solid supports in each of the pools. After the reaction, the pools of solid supports are combined, mixed, and split again. Each split pool is subjected to a second reaction or randomization that again is different for each of the pools. The process is continued until a library of target compounds is formed. Split/pool synthesis is a very efficient method that allows synthesis of a library of n1xc3x97n2xc3x97n3 members with just n1+n2+n3 reactions. Split/pool combinatorial synthesis is described further in, e.g., Furka and Bennett (1999) xe2x80x9cCombinatorial libraries by portioning and mixing,xe2x80x9d Comb. Chem. High Throughput Screening 2:105-122 and Lam et al. (1997) xe2x80x9cThe xe2x80x98one-bead-one-compoundxe2x80x99 combinatorial library method,xe2x80x9d Chem. Rev. 97:411-448.
The relative simplicity of split/pool synthesis is achieved at the expense of losing information about the identity of individual compounds during synthesis. As a consequence, structural determinations of synthesized compounds are typically performed following synthesis. Two general categories of techniques have been developed to identify the structures of individual library members during mixture deconvolution, namely, coding and noncoding strategies. Coding methods provide structure determination for libraries through the reading of a code that represents unambiguously the series of steps that a given solid support was subjected to during synthesis. The coding entity may be chemical which relies on the iterative coupling of chemical tags (e.g., peptides, oligonucleotides, isotopes, binary molecule systems, or the like) to orthogonally functionalized beads during library synthesis, where the tag structure is read using various analytical techniques. See, e.g., Czarnik (1997) xe2x80x9cEncoding methods for combinatorial chemistry,xe2x80x9d Curr. Opin. Chem. Biol. 1:60-66 and Barnes et al. (1998) S. Rec. Res. Dev. Org. Chem. 2:367-379. Various nonchemical encoding techniques have also been developed which record the synthetic or chemical history of library members by physical methods. See generally, Xiao and Nova (1997) Comb. Chem. 135-152. These techniques include, e.g., radiofrequency encoding (see, e.g., Nicolaou et al. (1995) Angew. Chem. Int. Ed. Engl. 34:24-2479 and Moran et al. (1995) J. Am. Chem. Soc. 117:10787-10788), and optical or color encoding (see, e.g., Xiao et al. (1997) Angew. Chem. Int. Ed. Engl. 36:780-782), where solid-phase supports are encapsulated in an encoded porous container.
Noncoding methods of determining compound structure involve techniques which do not utilize additional encoding constructs associated with library members structures. These methods include, e.g., synthesis in a fixed array (parallel synthesis), where a compound""s position within the array identifies the series of synthetic steps used to create the compound; direct deconvolution by pooling methods, where deconvolution of active structure is performed through selection of active pools from various synthetic cycles; and direct deconvolution by bioanalytical methods, where the chemical structure of active library components is determined by bioanalytical methods. See, e.g., U.S. Pat. No. 5,143,854 xe2x80x9cLARGE SCALE PHOTOLITHOGRAPHIC SOLID PHASE SYNTHESIS OF POLYPEPTIDES AND RECEPTOR BINDING SCREENING THEREOF,xe2x80x9d issued Sep. 1, 1992 to Pirrung et al., Pirrung (1997) xe2x80x9cSpatially addressable combinatorial libraries,xe2x80x9d Chem. Rev. 97:473-488, DeWitt et al. (1993) xe2x80x9cxe2x80x98Diversomersxe2x80x99: an approach to nonpeptide, nonoligomeric chemical diversity,xe2x80x9d Proc. Natl. Acad. Sci. USA 90:6909-6913, Geysen et al. (1986) xe2x80x9cA priori delineation of a peptide which mimics a discontinuous antigenic determinant,xe2x80x9d Mol. Immunol. 23:709-715, and Geysen et al. (1987) J. Immun. Meth. 102:259-274 (parallel synthesis of peptides on rods or pins).
Both coding and noncoding approaches to determining the identity of structures following split/pool synthesis have significant disadvantages. Although encoding strategies allow the use of the most efficient form of split/pool synthesis, pooling of solid phase synthesis units during intermediate steps in synthesis, encoding inevitably records only the series of steps that the support was exposed to during synthesis, which should, but does not necessarily, lead to the desired products. Furthermore additional steps are often required during synthesis and decoding to assign structure accurately. Parallel synthesis does not allow the most efficient means of synthesis as, by design, the support is split and not pooled during synthesis in order to unambiguously predict a structure to be present at a location in an array of compounds. Finally, the time and labor intensive nature of the processes employed to decode the synthetic product limit the application of this method to a small portion of the total number of synthetic productsxe2x80x94typically, those structures which show activity in high throughput screening assays. In this approach, valuable information about closely related but inactive structures is not obtained.
Combinatorial chemistry has advanced to the point that it is not enough to synthesize a desired set of compounds. It has now become equally important to consider the steps that immediately follow synthesis. For example, within the last several years there is a clear trend in combinatorial chemistry towards producing pure, characterized individual compounds. Consequently, compound analysis, to assess purity and confirm that the intended compounds were synthesized, is routinely conducted following synthesis of combinatorial libraries. Mass spectrometry (MS) is usually the method used for confirmation of structure. High performance liquid chromatography (HPLC) is most often used for purity assessment. Typically, components of a combinatorial library are subjected to HPLC/MS analysis for quality control which is independent of the way in which the relevant library was synthesized (by parallel synthesis, encoding, etc). Also, the format of the screening assays, which will be used for testing compounds originating from combinatorial synthesis, is relevant.
From the above, it is apparent that there is a substantial need for new methods which permit more efficient production of large libraries of compounds with easily identifiable structures by combinatorial synthesis and that enable all steps, including synthesis, analysis, and screening, to be performed as efficiently as possible. The present invention provides new methods, and related systems, for efficiently synthesizing and identifying structural features of combinatorial library members. These and a variety of additional features will become evident upon complete review of the following.
The present invention provides a method of identifying selected members of a synthesized library of materials, which in several embodiments is completely or partially computer implemented. The methods relate to synthetic strategies which use synthetic pooling strategies and, e.g., data analysis which accounts for shared chemical histories of products of the synthetic strategies, to determine unambiguously the structures of the synthesized products.
The methods typically include, e.g., (a) providing at least n*m*f solid phase synthesis units in which n is equal to a number of choices of different first components in a first stage of synthesis, m is equal to a number of choices of different second components in a second stage of the synthesis, and f is equal to a number of solid phase synthesis units to include identical materials upon completion of the synthesis. The method also typically includes (b) segregating the solid phase synthesis units into n separate first stage reaction vessels in which each separate first stage reaction vessel comprises at least m*f solid phase synthesis units and (c) reacting the solid phase synthesis units in each of the separate first stage reaction vessels with a different first component in the first stage of the synthesis. Thereafter, the method typically includes (d) segregating the solid phase synthesis units of (c) into m separate second stage reaction vessels by distributing at least one of the solid phase synthesis units from each of the separate first stage vessels into each separate second stage reaction vessel such that each of the separate second stage reaction vessels comprises at least n*f solid phase synthesis units and (e) reacting the solid phase synthesis units in each of the separate second stage reaction vessels with a different second component in the second stage of the synthesis to synthesize the library of the materials (e.g., producing a combinatorial chemical library or the like). The method additionally includes (f) detecting one or more distinguishing physical properties (e.g., different molecular masses or the like) of selected members of the library and (g) identifying the selected members based on the one or more detected distinguishing physical properties. Data deconvolution which takes advantage of an understanding of the shared chemical histories of the various solid phase supports during synthesis can be used to assign structures to the various library members based upon the distinguishing physical property or properties.
In some embodiments, the at least n*m*f solid phase synthesis units are subjected to one or more split/pool synthesis steps prior to (a). Thus, (a) optionally includes (i) segregating the at least n*m*f solid phase synthesis units into p separate third stage reaction vessels in which p is equal to a number of choices of different third components in a third stage of the synthesis, and in which each separate third stage reaction vessel comprises at least n*m*f/p solid phase synthesis units. In certain embodiments, the at least n*m*f solid phase synthesis units include n*m*f*p solid phase synthesis units. In this embodiment, (a) also optionally includes (ii) reacting the solid phase synthesis units in each of the separate third stage reaction vessels with a different third component in the third stage of the synthesis and (iii) combining and mixing the solid phase synthesis units of (ii) in a single pool to provide the at least n*m*f solid phase synthesis units. Optionally, this embodiment further includes (iv) separating the at least n*m*f solid phase synthesis units of (iii) into n*m separate containers in which the n*m separate containers are segregated into the n separate first stage reaction vessels as the solid phase synthesis units of (b). As an additional option, this embodiment further includes separating the at least n*m*f solid phase synthesis units of (c) into n*m separate containers in which the n*m separate containers are segregated into the m separate second stage reaction vessels as the solid phase synthesis units of (d).
In certain embodiments, (f) further comprises cleaving the materials from the solid phase synthesis units prior to detecting the one or more distinguishing physical properties (e.g., different molecular masses or the like). In other embodiments, the solid phase synthesis units of (e) each include multiple particles combined together, and (f) further includes separating selected particles from other particles and cleaving synthesized materials from the selected particles prior to detecting the one or more distinguishing physical properties. The different molecular masses are detected, most typically, by mass spectrometry. In certain embodiments, structural identification of the selected members includes subtracting a mass of the different second component reacted in a particular separate second reaction vessel from the different detected masses of the selected members to determine masses of different first components included in each of the selected members. The structural identification typically accounts for mass defects of reaction of the selected members. Optionally, structural identification of the selected members includes determining a fingerprint of library members in one or more of the separate second stage reaction vessels.
In preferred embodiments, structural identification of the selected members includes correlating the different detected masses of the selected members to a physical or logical matrix that includes masses for each individual library member. The correlation is generally computer implemented. For example, at least one entry in the matrix includes a summation of masses of different combinations of first and second components. Some or all entries in the matrix can be summations of different combinations of first and second components, and optionally, of other components (e.g., third components or the like). Furthermore, correlations of the different detected masses to entries in the matrix typically account for mass defects of reaction of the selected members (mass differences between predicted and observed masses).
The present invention also relates to a combinatorial library synthesis system that includes (a) a plurality of reaction vessels, (b) a handling system (including, e.g., a bead handler or the like) configured to move solid phase synthesis units and reagents to and from the plurality of reaction vessels, (c) a detection system (e.g., a mass spectrometer or the like) to detect one or more distinguishing physical properties (e.g., different masses or the like) of selected members of the combinatorial library, and (d) a computer operably connected to the handling and detection systems. The computer can include system software which directs the handling or detection systems to: (i) segregate the solid phase synthesis units into n separate first stage reaction vessels to provide m*f solid phase synthesis units in each of the n vessels in which n is equal to a number of choices of different first components in a first stage of a library synthesis, m is equal to a number of choices of different second components in a second stage of the library synthesis, and f is equal to a number of solid phase synthesis units which comprise identical materials on completion of the library synthesis. The system software can also direct the handling or detection systems to: (ii) deliver one or more of the different first components to each of the n separate first stage reaction vessels to provide for reaction of the different first components with the solid phase synthesis units to provide first stage reacted solid phase members and (iii) segregate the first stage reacted solid phase members from the n separate first stage reaction vessels into m separate second stage reaction vessels by distributing at least one of the first stage reacted solid phase members from each of the separate first stage reaction vessels into each second stage reaction vessels such that each second stage reaction vessel comprises at least n*f solid phase synthesis units. Separately or in addition, the system software can also direct the handling or detection systems to: (iv) deliver one or more different second components to the second stage reaction vessels to provide for reaction of the different second components with the first stage reacted solid phase members to provide the combinatorial library and (iv) detect one or more distinguishing physical properties (e.g., different masses or the like) of the selected members of the combinatorial library. The system software also typically directs the handling system in (iv) to effect cleavage of combinatorial library members from the solid phase synthesis units.
In some embodiments prior to (i), the system software directs the handling system to: (1) segregate at least n*m*f solid phase synthesis units into p separate third stage reaction vessels in which p is equal to a number of choices of different third components in a third stage of the library synthesis, and in which each separate third stage reaction vessel comprises the at least n*m*f/p solid phase synthesis units and (2) deliver one or more of the different third components to each of the separate third stage reaction vessels to provide for reaction of the different third components with the solid phase synthesis units to provide third stage reacted solid phase members, and (3) combine and mix the third stage reacted solid phase members in a single pool to provide the solid phase synthesis units for (i). In some embodiments, the system software further directs the handling system to: (4) separate the solid phase synthesis units of (3) into n*m separate containers in which the n*m separate containers are segregated into the n separate first stage reaction vessels as the solid phase synthesis units of (i). Optionally, the system software further directs the handling system to separate the solid phase synthesis units of (3) into n*m separate containers in which the n*m separate containers are segregated into the m separate second stage reaction vessels as the solid phase synthesis units of (iii). As an additional option, each of the n*m separate containers comprises multiple particles combined together.
In preferred embodiments, the computer further includes at least one database having a logical matrix corresponding to masses of members of a virtual library that are correlated with the detected masses of the combinatorial library members produced by the system to thereby identify chemical structures of the combinatorial library members. Correlations typically account for mass defects of reaction. At least one entry in the logical matrix typically includes a summation of masses of different combinations of first and second components.
The invention can also be embodied in kits, e.g., including any of the system elements for performing any of the methods herein, and optionally further including containers for holding any of the relevant system elements, packaging materials, instructional materials for practicing the method, and the like.